The iron export protein ferroportin is an essential regulator of systemic iron homeostasis. Following uptake by enterocytes, dietary iron can be effluxed into the plasma via the actions of ferroportin, a membrane bound iron transporter, to the basolateral side of intestinal epithelial cells. Ferroportin is also expressed on the cell surface of hepatocytes and macrophages where it plays a key role in releasing stored iron and iron recycled from phagocytosed erythrocytes, respectively (Andrews, et al. Annu. Rev. Physiol. 69 (2007), 69-85). Ferroportin stability is posttranslationally regulated by the hormone hepcidin, a circulating member of the defensin family of peptides that is secreted primarily by the liver (Andrews, et al. Blood 112 (2008), 219-230). When serum iron levels are sufficient to satisfy systemic demand, the gene encoding hepcidin (HAMP) is upregulated and hepcidin is secreted into the blood where it binds ferroportin (Nicolas, et al. J. Clin. Invest. 110 (2002), 1037-1044). This interaction induces structural changes within ferroportin that promotes its internalization and degradation, diminishing iron export to effectively reduce serum iron levels (Nemeth, et al. Science 306 (2004), 2090-2093). Transcription of the hepcidin gene is attenuated when systemic demand for iron is high, ultimately stabilizing ferroportin and increasing iron efflux into the serum (Nemeth, et al. Science 306 (2004), 2090-2093; Drakesmith, et al. Blood 106 (2005), 1092-1097; and Pietrangelo, Blood Cells Mol. Dis. 32 (2004), 131-138).
A number of proteins are employed by hepatocytes to integrate various cues indicating systemic iron demands and to signal appropriate responses in hepcidin expression. Mutations to these components can result in impaired hepcidin expression and constitutively elevated levels of ferroportin, leading to primary hemochromatosis. For example, unchecked iron absorption in the duodenum can eventually result in iron overload, characterized by the toxic accumulation of iron in tissues such as the liver (Barton, Am. J. Med. Sci. 346 (2013), 403-412; Crownover, et al. Am. Family Physician 87 (2013), 183-190). In addition, iron overload can also result from another disease or condition (secondary hemochromatosis). The disease and condition include but are not limited to certain types of anemia, such as thalassemias and sideroblastic anemia; atransferrinemia and aceruloplasminemia; and chronic liver diseases, such as chronic hepatitis C infection, alcoholic liver disease, or nonalcoholic steatohepatitis. Other factors can also lead to secondary hemochromatosis, including but are not limited to blood transfusions, oral iron pills or iron injections (with or without very high vitamin C intake), and long-term kidney dialysis.
The human hemochromatosis gene, or HFE, encodes a membrane protein employed by the liver to sense serum iron levels. When high transferrin-bound iron levels are recognized by transferrin receptor 2 (TfR2), it recruits HFE protein to engage a BMP/SMAD-dependent signaling pathway to induce HAMP transcription. Recent work suggests that common polymorphisms to the HFE gene (e.g., leading to HFE protein with C282Y/C282Y homozygous, C282Y/H63D heterozygous, or C282Y/S65C homozygous mutation) compromise HFE's ability to affect BMP/SMAD signaling (Wu, et al. Blood 124 (2014), 1335-1343). Consequently, hepcidin expression is impaired and individuals homozygous or heterozygous for these missense alleles can develop hemochromatosis accompanied by damage to the liver and other organs resulting from iron overload. This disease can by modeled in mice through targeted inactivation of the HFE gene (e.g., HFE−/− mice; reviewed in Fleming, et al. Annu. Rev. Nutr. 31 (2011), 117-137). Some hemochromatotic patients can be treated by phlebotomy. However, some patients with forms of secondary hemochromatosis may not be treated by phlebotomy due to low red cell mass. These patients are dependent on iron chelators to treat the iron overload. While iron chelators are somewhat effective, the treatment is associated with significant adverse side effects (Maggio, Br. J. Haematol. 138 (2007), 407-421).
Hypoxia Inducible Factors 1α and 2α (HIF-1α and HIF-2α) are heterodimeric transcription factors whose regulated alpha subunits can be induced under low oxygen or iron conditions to upregulate hundreds of potential downstream target genes (Keith, et al. Nature Rev. Cancer 12 (2012), 9-22). Genetic approaches with mice lacking HIF-2α expression in the duodenum have indicated that the HIF-2α isoform selectively regulates several genes critical for intestinal iron absorption. These targets include ferroportin, as well as the duodenal cytochrome B (DcytB) that reduces iron in the lumen so that it may be transported into enterocytes through the divalent metal transport protein 1 (DMT-1), also a HIF-2α target. Mice lacking intestinal HIF-2α expression, absorb less iron and are resistant to the consequences of mutations that would otherwise promote hemochromoatosis (Mastrogiannaki, et al. J. Clin. Invest. 119 (2009), 1159-1166; Mastrogiannaki, et al. Blood 119 (2012), 587-590; and Anderson, et al. Proc. Natl. Acad. Sci. U.S.A. 26 (2013), 4922-4930).
Therefore, there is a need to develop new therapies to treat patients suffering from iron overload disorders and related diseases. Small molecule inhibitors of HIF-2α may fulfill the need and offer other advantages over the existing therapies.